Method for the acylation of the 7-amino group of the cephalosporanic ring

ABSTRACT

A method for the acylation of the 7-amino group of the cephalosporanic ring, according to which a 7-ACA amino thiazolyl protected adduct is prepared by acylating said amino group by an aminothiazolyl acetic acid whose amino group has been protected by a phenyl acetyl or a phenoxy acetyl group, the amino group being then deprotected by aqueous hydrolysis in the presence of penicillin G amidase or respectively penicillin V amidase.

The present invention relates to a method for the acylation of the7-amino group of the cephalosporanic ring.

7-ACA (7-amino-3-acetoxymethyl-3-cephem-4-carboxylic acid) of formula##STR1## is a well known compound which has been proposed as startingmaterial in various syntheses, in particular in the syntheses of manycephalosporins.

A number of the most important cephalosporins are obtained through thefollowing reaction steps:

a) effecting an acylation of the 7-amino group of the cephalosporanicring by an optionally substituted aminothiazolyl acetic acid whose aminogroup has been protected;

b) deprotecting the protected amino group; and

c) optionally substituting the 3-acetoxymethyl group of thecephalosporinic ring by a nucleophilic agent.

The sequencing of the above reported steps can be varied at will. Forinstance, the step sequencing can be a), b), c); or a), c), b); or c),a), b), too, as it is described in the Journal of Antibiotic, December1978; 1262-1271 and in the BE-A-823861 by Takeda (concerning theantibiotic CEFOTIAM).

In ever case, the acylation off the 7-amino group of the cephalosporinicring is carried out with an optionally substituted aminothiazolyl aceticacid whose amino group has been protected, the amino group being thendeprotected.

The known protecting groups usable in the practice require the use ofexpensive starting materials (such as trityl and BOC), criticalconditions for introducing the reactants and critical acid conditionsfor removing the protection.

The creation of the aminothiazol ring directly on the adduct has theshortcoming that it requires the use of highly dangerous reactants suchas diketenes and anhydrides.

It has been now surprisingly found that some extremely importantadvantages are attained if the amino protecting group is a phenyl acetylgroup or a phenoxy acetyl group: these protecting groups are cheap, theyare easily introduced, and compatible with the carbonyl promotingconditions which are necessary for the reaction with 7-ACA.

The use of phenyl- or phenoxy-acetic acids to protect the amino group ofthe amino thiazolyl acetic acid (in order to promote the carboxylicgroup thereof) is extremely important because such a protection providesa very stable adduct extremely difficult to be chemically eliminated, sothat said adduct, because of such protection, can be subjected to thesubsequent chemical treatments without involving the protecting group.

Further, it has been surprisingly found that the above mentionedprotecting groups are selectively removable under extremely mildconditions, by simple hydrolysis in aqueous solution substantially atroom temperature in the presence of penicillin G amidase or penicillin Vamidase which catalyze the hydrolysis of the amidic bond of the N-phenylacetyl or N-phenoxy acetyl amino thiazolyl moiety at a much higher speedthan the one with which it hydrolyzes the amidic bond that is present inthe position 7 of the final compound of the reaction.

The penicillin G amidase and penicillin V amidase enzymes are known perse and their use has been described, for instance, in GB-A-1480850 andGB-A-1473100: by making use of such enzymes it was known to obtain byenzymatic route (in the place of the chemical route) 6-APA starting frompencillin G or penicillin V, and 7-ADCA starting from cephalosporin G orcephalosporin V.

Therefore the use of the N-phenyl- or N-phenoxyacetic protecting groupselectively removable by enzymatic hydrolysis in aqueous solution,without either removing the acetic ester group present in the moleculeor significantly hydrolyzing the amidic bond in 7, represents a veryimportant economical and ecological advantage with respect to themethods for acylating the 7-amino group of the cephalosporanic ring, inparticular for protecting and deprotecting the amino group in the knownsyntheses of 7-aminothioazolyl cephalosporins.

Of particular important is also the fact that the reaction occurs in anaqueous environment and high yields are obtained.

Consequently, the present invention concerns a method for the acylationof the 7-amino group of the cephalosporanic ring, wherein a 7-ACA aminothiazolyl protected adduct is prepared by acylating said amino group byan optionally substituted aminothiazolyl acetic acid whose amino grouphas been protected, the protected amino group being thereafterprotected, characterized in that the amino protecting group is selectedfrom the group consisting of a phenyl acetyl and respectively of aphenoxy acetyl group and that the deprotection is effected by hydrolysisin aqueous solution at a temperature of from 0° C. to 50° C. and at a pHfrom 5 to 9 in the presence of an enzyme selected from the groupconsisting of penicillin G amidase and penicillin V amidaserespectively.

In particular, it has been found that said hydrolysis may be effected ata temperature of from 15° C. to 35° C. and at a pH from 6 to 8.

The invention concerns also the novel amino thiazolyl acetic acid andits α-substitution derivatives whose amino group is protected by aphenyl carbonyl group selected from the group consisting of a phenylacetyl and phenoxy acetyl group; and also the 7-ACA amino thiazolylprotected adduct, where in the protection of the amino group made by aphenyl carbonyl group selected from the group consisting of a phenylacetyl and phenoxy acetyl group.

Of course, also the novel N-phenylacetyl and N-phenoxyacetyl optionallyα-aminothiazolyl acetic acid substituted and 3-substitutedcephalosporins form part of the present invention.

In order to make the features of the present invention clear, some nonlimitative embodiments thereof will now be detailedly described.

The following non limitative examples will illustrate the invention.

EXAMPLE 1 PREPARATION OF THE 7-[2(AMINOTHIAZOL-4-YL)]ACETAMIDOCEPHALOSPORANIC ACID A) Preparation of N-phenylacetyl amino thiazolylacetic acid (protected acylating)

18,6 g (0,1 moles) of the ethyl ester of amino thiazolyl acetic acid in100 ml of organic non hydroxylated solvent (methylene chloride, THF,dioxane, acetonitrile etc.) in the presence of 1,2 molar equivalents ofan organic base as triethylamine are added under stirring at 0° to 5° C.with 15,4 g (0,1 moles) off phenyl acetic acid chloride within 15minutes.

The reaction is complete after some hours at room temperature (TLC onsilica in ethyl acetate/hexane 7:3 Rf: about 0,7). The vessel is thencooled under ice, and the organic phase is then washed in sequence withdiluted hydrochloric acid, bicarbonate aqueous solution and water. Thesolution is dried and then evaporated under vacuum. The oily residuesolidifies spontaneously. The product can be crystallized byhexane/ethyl aetate. However this is not necessary because the rawproduct can be used directly for the next step. Said step consists intreating the solution of the above obtained ester with a NaOH aqueoussolution, said solution being obtained dissolving 1 g of NaOH every 4 gof ester to be hydrolyzed. A slight heating is produced when thesolutions are admixed. The reaction is kept stirred for 5 to 6 hours, atime sufficient to effect hydrolysis, whose development is followed byTLC.

When the reaction is complete, the mixture is evaporated under vacuum toeliminate most of solvent, maintaining the boiling temperature low, soto avoid the amidic bond hydrolysis. The reaction mixture is thendiluted with water, under stirring and the cooled and acidified withHCl. The precipitate consisting of the N-phenylacetyl amino thiazolylacetic acid (hereinafter called compound "X") is collected by filtrationand then washed with water on the filter and finally dried giving 22 g(79%).

B) Preparation of the 7-amido-((2-N-phenylacetylamino-4-thiazolyl)-2-acetyl)-3-acetoxymethyl-3-cefem-4-carboxylic acid

13,8 g (0,05 mole of the acid "X" obtained according to A abovesuspended in 30 ml of methylene chloride are treated with 9,5 g ofoxalyl chloride (0,075 mole) and with some drops of DMG at roomtemperature, under stirring. When the gas development has practicallyceased, 3 g further of oxalyl chloride are added, heating under slightreflux for 30 minutes. The reaction is then concentrated under vacuum.The residue, having an intense green colour, is admixed with 50 ml ofmethylene chloride and is added dropwise, under stirring and undernitrogen atmosphere at -10° C., to a mixture of 12,2 g (0,045 mole) of7-ACA and 40 ml of triethylamine in 100 ml of methylene chloride.

When the addition is ended, the temperature is allowed to rise to roomtemperature and then the reaction mixture is washed with cool water anddiluted hydrochloric acid.

The dried organic phase is evaporated to give an oily red colouredresidue. This material, well dried under vacuum, may solidifyspontaneously.

However, if it is admixed with a little volume of ethanol at 50° to 60°C., it permits the separation of a crystalline solid (compounds "Y"),which is collected by filtration. 16,7 g (67%). Similar yields areobtained when treating the 7-ACA suspension in methylene chloride with 2molar equivalents of BSA, till complete dissolution, followed by theaddition of triethylamine and then of the acid chloride.

Alternatively, the same hereabove specified compound can be obtained asfollows:

13,8 g (0,05 mole) of the acid obtained according to A above in 300 mlof THF in the presence of 6,2 g (0,06 mole) of N-methyl morpholine areadded dropwise with 5,4 g of ethyl cloroformate. After 3 hours, 13,6 g(0,05 mole) of 7-ACA in 50 ml of methylene chloride and 50 ml oftriethylamine are added at room temperature.

After 12 hours, always at room temperature, the reaction mixture isconcentrated under vacuum, then diluted with methylene chloride andrepeatedly washed with diluted HCl and water and finally dried. Thereddish residue obtained by evaporating the solvent is then treated withethanol, thus obtaining a solid 7-ACA amino thiazolyl protected adduct(compound "Y") which is filtered. 14 g (59%).

C) Enzymatic Hydrolysis

5.3 g of the compound "Y" obtained according to B above are suspended in120 ml of water and the suspension is treated with NaOH 1N at pH 8, at36° C. All the solid is dissolved within 10-15 minutes. 500 units of thePGA enzyme are then added and the hydrolysis development is followed byHPLC, because no direct correspondence between the consumed base and thehydrolysis evolution is observed. When the starting material hasdisappeared, the immobilized enzyme is filtered, the reaction mixture isconcentrated under vacuum, at a volume of 50 ml, then it is cooled andacidified to pH 3,6. The precipitate is collected and washed off thefilter with absolute ethanol. The product is the7-[2(aminothiazol-4-yl)]acetamido cephalosporanic acid which can becrystallized from aqueous ethanol. 3,3 g (80%).

EXAMPLE 2 Preparation of the7[((2-amino-4-thiazolyl)acetyl)amino]-3-[[[1-[2-(dimethyl-amino)-ethyl]-1H-tetrazol-5-yl]-thio]methyl-8-oxo-5-thia-1-aza-bicyclo[4,2,0]oct-2-ene-2-carboxylicacid 2HCl

(CEFOTIAM)

5 g (12,1 mmoles) of the 7-[2(aminothiazol-4-yl)]acetamidocephalosporanic acid obtained according to Example 1-C above aredissolved in 40 ml of H₂ O with 2,3 g (13.2 mmoles) of1-(2-dimethyl-aminoethyl)-1H-tetrazole-5-thiol (compound "Z") and 2,18 g(26 mmoles) of NaHCO₃ at 70° C. The mixture is permitted to react at 70°C. for 2 h following the reaction by TLC.

The mixture is then cooled at 20° C. and the solution, at pH 7, iseluted on XAD 1180.

The product is eluted with a H₂ O/methanol mixture, evaporating undervacuum the enriched faction and cefotiam is crystallized by addingisopropanol and HCl concentrated. Filtering and drying under vacuum arethen effected.

Molar yield: 67%.

Alternatively:

20 mmoles of the 7-[2-(aminothiazol-4-yl)]acetamido cephalosporanic acidof Ex. 1-C are treated with 20 mmoles of the above mentioned compound"Z" in 150 ml di THF/H₂ O 1/1 and 40 mmoles of NaHCO₃ at 60° C. under N₂for 4 hours.

At the end of the reaction, the solution is concentrated under vacuum,adjusting the pit at 7, and extracted for 3 times with 40 ml of CH₂ Cl₂.The residual aqueous suspension is further concentrated and acidified atpH 3,0 with concentrated HCl, then it is cooled at 0° C. and filtered.The solid is purified in isopropanol at 50° C. for 30' and then cooled,filtered and dried.

Molar yield: 58%.

EXAMPLE 3 PREPARATION OF CEFOTIAM A) Preparation of the7-amino-ceph-3-em-3-1-(2-dimethyl-aminoethyl)-1H-tetrazol-5-thiol-4-carboxylicacid.

15,3 g (88,3 mmoles) of 1-(2-dimethyl-aminoethyl)-1H-tetrazole-5-thiol(compound "Z") are added in 100 ml of trifluoroacetic acid (TFAA) cooledat -5° C. Then 24 g of 7-ACA (88,1 mmoles) are charged, in littleamounts, in 30' at a temperature from 0° to -5° C. The disappearing ofthe 7-ACA is followed by TLC. At the end of reaction, TFAA is evaporatedunder vacuum at 45° C. 50 ml of THF are added to the concentrate.

The precipitate attained is then filtered under vacuum. The solid isrecovered with H₂ O at pH 7 and with NaHCO₃ to release the base of thetitle compound as a white solid which is filtered under vacuum anddried.

Yield: 76%

Alternatively:

15,3 g (88,3 mmoles) of the above mentioned compound "Z" are added in100 ml of methansulphonic acid cooled at +5° C. 24 g of 7-ACA (88,1mmoles) are then charged in little amounts at a temperature from 0° to+5° C.

At the end of the reaction, 100 ml of isopropilic acid are added tocrystallize the dimethansulphonate salt of the title compound.

Filtering is then effected and the solid is set in 80 ml of H₂ Oadjusting to pH 7 with NaHCO₃. 30 ml of isopropanol are added and thecrystallization of the title compound is effected at 15° C.

Yield: 78%

NMR (δ in DMSO-d6):

2,58 (6H, s, Ndimethyl); 3,22 (2H, t, etero Nmethylen); 3,52 (2H, q AB,2CH₂); 4,24 (2H, q, 3CH₂); 4,60 (2H,m, CH₂ -Ndimethyl); 4,76 (1H, d,6CH); 4,94 (1H, d, 7CH).

B ) Preparation of a N-phenylacetyl protected 3 -substitutedcephalosporin

10 mmoles of the protected acylating agent "X" in 10 ml of anhydrous DMFand 20 ml of CH₂ Cl₂ are treated with 10 mmoles of N-methylmorfoline at-30° C. 10 mmoles of ethyl chloroformate are then added maintaining thetemperature at -30° C. for 30'. The mixture is then poured in a 10mmoles solution of the compound obtained according to A) above,dissolved in 10 ml of DMF, 20 ml of CH₂ Cl₂ and 10 mmoles of N methylmorfoline. The condensation reaction is complete after 1 hour; thesolvent is evaporated under vacuum and the residual is recovered with alittle of water and adjusted to pH 3,5 with concentrated HCl. Acrystalline solid is then filtered and purified in isopropanol at 4° C.

The molar yield is 65%.

Alternatively:

20 mmoles of the protected acylating agent "X" are reacted with 20mmoles of the compound obtained according to A) above, in 200 ml or CH₃CN/H₂ O 1/1 and 50 mmoles of NaHCO₃ at 65° C. for 3 hours under N₂ inertatmosphere.

At the end of the reaction, concentration under vacuum is effected,adjusting the pH at 7,0 and then effecting two extractions with 45 ml ofCH₂ Cl₂. The aqueous suspension is then further concentrated andadjusted to pH 3,5 and filtered.

The solid can be subjected to the enzymatic hydrolysis as it is or itcan be purified under heating in isopropilic alcohol, filtered anddried.

Yield: 55%

NMR (δ in DMSO-d6):

2,62 (6H, s, Ndimethyl); 3,40 (2H, t, etero Nmethylen); 3,50 (2H, q AB,2CH₂); 3,65 (2H, s, CH₂ --Ph); 3,76 (2H, s, CH₂ --CO.sub.); 4,25 (2H, qAB,3CH₂); 4,65 (2H,m,CH₂ -Ndimethyl); 5,05 (1H,d,6CH); 5,65 (1H,q,7CH);6,92 (1H,s,thiazole 5H); 7,30 (5H,m,Ph).

C) Enzymatic Hydrolysis

10 g (15,5 mmoles) of the N-phenylacetyl protected cephalosporinobtained as per B) above are dispersed into 100 ml of H₂ O at roomtemperature, and then taken back into solution with soda 1N at pH 7,5.

10 g of PG Amidase immobilized on Eupergit C (170 IU/g) are added tothis solution following the enzymatic hydrolysis with NaCH1N by anautomatic titrator.

The base consuming is noted to correspond to the formation of cefotiam(also followed in TLC). The enzyme is filtered when the reaction isended and the solution is passed on an adsorbing resin XAD 1180.

The eluate of the resin which contains cefotiam is exsiccated, collectedwith HCl 4N and added with isopropanol to crystallize cefotiamdihydrochloride. Filtering and drying under vacuum are then effected.

Molar yield: 72%

EXAMPLE 4 PREPARATION OF CEFOTAXIME A) preparation of the N-phenacetylamino thiazol α methoxyimino acetic

10 g of methoxyiminoaminothiazolacetic acid in 100 ml of methylenechloride and 20 ml of triethylamine are added at 0° C. under stirring to1,2 molar equivalents of the phenylacetic acid chloride. After somehours, 10 ml of triethylamine and further 0,5 equivalents of thechloride are further added. The reaction is complete after one night.The organic phase is washed with water, then dried and concentrated. Theresidual is slurried with a little of ethanol which removes an eventualresidual of phenylacetic acid.

Yield: about 80%.

1H NMR DMSO-d6

3,75 (2H,s,PhCH₂); 3,90 (3H,s,NOCH₃); 7,30 (5H,m,Ph); 7,50 (1H,s,SCH);12,75 (1H,s,COOH).

B) Preparation of the N-phenylacetylcefotaximina

2 g of the product obtained according to the Example 4-A in 60 ml ofanhydrous THF are added at about 5° C. with 0,65 g ofdicyclohesylcarboimide, corresponding to 0,5 molar equivalents. Themixture is kept under stirring at said temperature for 30 minutes, thenfor further 30 minutes at room temperature.

The precipitate of dicyclohesylurea formed in the midtime is filteredand the organic phase is cooled at -20° C.

A solution of 0,85 g of 7-ACA in 15 ml of methylene chloride and 2 ml oftriethlylamine is added therewith in the space of some minutes. Thetemperature is then allowed to raise, so maintaining it for 3 hours. Thesolution is evaporated and then the residue is recovered with dioxane,said residue consisting of a mixture of N-phenacetylcefotaxime and ofN-phenacetyl amino thiazol α-methoxyimino acetic thiazolacetic acidprecipitating with diethylamine said last compound as a salt. Filteringdrying are then effected. The residue is crystallized from ethyl acetateto give the desired N-phenacetylcefotaxime.

Alternatively, the separation can be effected digesting the residueconsisting of the mixture of the two above mentioned compounds with alittle of methylene chloride wherein the N-phenacetyl amino thiazolα-methoxy-imino acetic acid is less soluble, and then crystallizing theresidue after evaporating CH₂ Cl₂ from ethyl acetate.

Molar yield: 71%

1H-NMR DMSO-d6 2,05 (3H,s,COCH₃); 3,55 (2H,qAB,2CH₂); 3,75 (2H,s,PhCH₂);3,90 (3H,s,NOCH₃); 4,65-5,0 (2H,qAB,3CH₂); 5,18 (1H,d,6CH); 5,85(1H,q,7CH); 7,3 (5H,m,Ph); 7,4 (1H,s,SCHC); 9,75 (1H,d,CONH); 12,8(1H,S,COOH).

C) Enzymatic Hydrolysis

0,5 g of N-phenacetylcefotaxime are dissolved in 2 ml of acetonitrileand added, under stirring, to 25 ml of a buffer at pH 8.

About 400 UI of PGA on Eupergit C are added and the course of thehydrolysis is followed by TLC at 37° C. The reaction is ended in 40-50minutes. Filtering and then concentrating in a very small volume areeffected, acidifying at 0° C. Cefotaxime is recovered by filtration andis then re-dissolved after cooling with a little of ethyl acetate andfiltered to remove the residue of the phenylacetic acid.

Molar yield: 65%

EXAMPLE 5

Carrying out the reaction as described in the Example 4, there has beenfound the possibility to produce other important cephalosporins, such asCEFTAZIDIME, CEFMENOXIME, CEFIXIME, CEFTRIAXONE, CEFODIZIME, CEFTIBUTEN,CEFTIZOXIME, CEFEPIME.

The 7-ACA amino thiazolyl protected adducts eventually 3-substitutedfrom which the cephalosporins derive, where the amino protecting groupis a phenyl acetyl group, taken into solution at pH 7,5, with or withoutCH₃ CN at 10%, have been subjected to the action of PGA whereas the pHhas been maintained constant with a base. There has been demonstratedhow, also in these cases, the PGA is selective, in its hydrolyticaction, as to the 7-amidic bond of cephalosporin. At the end of theenzymatic hydrolysis, PGA is collected by filtration, concentrated undervacuum, slightly acidified and extracted with an organic solventimmiscible with water: the phenyl acetic acid. The aqueous phase isconcentrated to a little volume and the above mentioned cephalosporinsare recovered.

Obviously, in the case of some cephalosporins, the acetic moiety on theside chain had been previously protected as t-butylic ester to be thenremoved in the final step by slightly acidic hydrolysis.

The same procedure as disclosed for the N-protected phenylacetic seriesis valid for effecting the protection of phenoxy acetic moieties, inwhich case penicillin V amidase is used.

We claim:
 1. A method for producing a 7-aminothiazolyl cephalosporaniccompound of formula (I): ##STR2## wherein R is selected from the groupconsisting of ##STR3## and X is selected from the group consisting ofhydrogen, ═N--O--CH₃, ═N--O--H, ═N--O--CH₂ --COOH, ##STR4## and--CH--CH₂ --COOH; comprising reacting the 7-amino group of a compound offormula (II): ##STR5## wherein R is defined as above, with the compoundof formula (III): ##STR6## wherein L is a phenylacetyl or phenoxyacetylgroup and X is defined as above, to form an N-protected aminothiazolylcephalosporanic compound of formula (IV): ##STR7## wherein R, L and Xare defined as above; hydrolysing said compound of formula (IV) with anenzyme selected from the group consisting of penicillin G amidase andpenicillin V amidase in an aqueous solution at a temperature of from 0°C. to 50° C. at a pH from 5 to 9 to provide said compound of formula(I).
 2. A method according to claim 1, characterized in that saidhydrolysis is effected at a temperature of from 15° C. to 35° C. and ata pH from 6 to 8.